Abstract

Deregulation of KRAS4b signaling pathway has been implicated in 30% of all cancers. Membrane localization of KRAS4b is an essential step for the initiation of the downstream signaling cascades that guide various cellular mechanisms. KRAS4b plasma membrane (PM) binding is mediated by the insertion of a prenylated moiety that is attached to the terminal carboxy-methylated cysteine, in addition to electrostatic interactions of its positively charged hypervariable region with anionic lipids. Calmodulin (CaM) has been suggested to selectively bind KRAS4b to act as a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway by displacing KRAS4b from the membrane. However, the mechanism by which CaM can recognize and displace KRAS4b from the membrane is not well understood. In this study, we employed biophysical and structural techniques to characterize this mechanism in detail. We show that KRAS4b prenylation is required for binding to CaM and that the hydrophobic pockets of CaM can accommodate the prenylated region of KRAS4b, which might represent a novel CaM-binding motif. Remarkably, prenylated KRAS4b forms a 2:1 stoichiometric complex with CaM in a nucleotide-independent manner. The interaction between prenylated KRAS4b and CaM is enthalpically driven, and electrostatic interactions also contribute to the formation of the complex. The prenylated KRAS4b terminal KSKTKC-farnesylation and carboxy-methylation is sufficient for binding and defines the minimal CaM-binding motif. This is the same region implicated in membrane and phosphodiesterase6-δ binding. Finally, we provide a structure-based docking model by which CaM binds to prenylated KRAS4b. Our data provide new insights into the KRAS4b-CaM interaction and suggest a possible mechanism whereby CaM can regulate KRAS4b membrane localization.

GDP- and GNP-bound KRAS4b-FMe binds to CaM in a nucleotide-independent manner. ( A…

Figure 2

GDP- and GNP-bound KRAS4b-FMe binds to CaM in a nucleotide-independent manner. (A) Shown are the sedimentation velocity and normalized absorbance c(s) profiles for solutions containing 42 μM CaM (green), 8 μM GDP-bound KRAS4b-FMe (red), and 10 μM GNP-bound KRAS4b-FMe (blue). C(s) distributions for mixtures of 20 μM KRAS4b-FMe and 40 μM CaM as well as 14 μM KRAS4b-FMe and 6 μM CaM are shown as long dash and short dash plots, respectively. Red plots are data for GDP-bound KRAS4b-FMe, and blue plots are for GNP-bound KRAS4b-FMe. (B and C) Shown are SPR binding kinetics of 10–0.2 μM GDP- and GNP-bound KRAS4b-FMe to avi-CaM. Data were fit and yielded Kd values of 0.4 and 0.5 μM, respectively. (D) Fits of the steady-state binding isotherms derived from the SPR data are shown.

Figure 3

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KRAS4b-FMe forms a 2:1 complex…

Figure 3

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KRAS4b-FMe forms a 2:1 complex with CaM. ( A ) Shown are the…

Figure 3

KRAS4b-FMe forms a 2:1 complex with CaM. (A) Shown are the absorbance (blue) and interference (red) Sw isotherms for the titration of CaM into 3 μM GDP-bound KRAS4b-FMe (left panel) and 6 μM GDP-bound KRAS4b-FMe (center panel) as well as the titration of GDP-bound KRAS4b-FMe into 6 μM CaM (right panel). Experimental data (circles) were fitted globally to a two-site equilibrium model with two nonsymmetric sites and microscopic K as described in the text to obtain the isotherms shown (solid lines). (B) MALS analysis was performed on GDP-bound KRAS4b-FMe-CaM complex eluted from an SEC column. The absolute molar mass versus elution time displays a monodisperse peak at 57 kDa.

Figure 4

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KRAS4b-FMe forms a 1:1 complex…

Figure 4

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KRAS4b-FMe forms a 1:1 complex with CaM-N and CaM-C. ( A ) Shown…

Figure 4

KRAS4b-FMe forms a 1:1 complex with CaM-N and CaM-C. (A) Shown are the absorbance (blue) and interference (red) Sw isotherms for the titration of CaM-C into 3 μM GDP-bound KRAS4b-FMe. Experimental data (circles) were fitted to an A + B = AB heteroassociation model to obtain the best-fit isotherms shown (solid lines). (B) Shown are the absorbance (blue) and interference (red) Sw isotherms for the titration of CaM-N into 3 μM GDP-bound KRAS4b-FMe. Experimental data (circles) were fitted to an A + B = AB heteroassociation model to obtain the best-fit isotherms shown (solid lines). Sedimentation velocity c(s) profiles for these isotherms are shown in Supporting Materials and Methods. (C) Shown are SPR binding kinetics of 10–0.2 μM GDP and GNP-bound KRAS4b-FMe to avi-CaM-C. Data were fit using a one-site model and yielded Kd values of ∼0.6 μM for both GDP- and GNP-bound KRAS4b-FMe to CaM-C. (D) Shown are SPR binding kinetics of 10–0.2 μM GDP- and GNP-bound KRAS4b-FMe to avi-CaM-N. Data were fit using a one-site model and yielded Kd values of ∼2 μM for both GDP- and GNP-bound KRAS4b-FMe to CaM-N. (E) Fits of the steady-state binding isotherms derived from the SPR data are shown.

Proposed structural docking model of CaM-KRAS4b interaction. (A) Cartoon showing two methylated and farnesylated KRAS4b subunits (PDB: 5TAR), color-coded magenta and cyan, modeled into two hydrophobic pockets of the pseudosymmetrical N- and C-terminal domains of calmodulin structure, color-coded green (PDB: 2MGU). The last five residues of KRAS4b (181–185) critical for binding to calmodulin and the hydrophobic residues in the hydrophobic pockets of calmodulin are shown in blue and yellow sticks, respectively. The farnesyl group covalently linked to the last residue of KRAS4b is also shown in sticks, having the same color as its respective KRAS protein subunit. (B) A surface representation of the structural model shown in (A) with the last residue of KRAS4b (185) and the farnesyl group displayed as sticks is given.